Project Summary Our novel studies show that the androgen receptor (AR) and the modulator of AR action co-repressor COUPTF-II/NR2F2 are co-expressed in theca cells of mouse ovarian follicles and in theca cells of normal cycling and PCOS patients. Exposure of mice to androgens in vivo leads to marked changes in theca cell morphology and gene expression profiles, most notable of which is the marked induction of vascular cell adhesion molecular 1 (Vcam1). Recent provocative evidence that disruption of AR selectively in mouse theca cells prevents an androgen-mediated PCOS phenotype and the induction of Vcam1 provides key evidence for critical actions of AR in theca cells. Theca cells propagated from PCOS women express higher levels of VCAM1 and CYP17A1 than normal theca cells, further supporting the notion that elevated VCAM1 and androgens contribute to altered ovarian functions and dysfunctions in PCOS and hyperthecosis. Based on these compelling observations, we hypothesize that elevated androgens alter AR regulation of theca cell functions leading to specific changes in cellular and molecular events, including the induction of Vcam1, that underlie and/or contribute to ovarian dysfunction in PCOS and hyperthecosis. We propose to use genetically- modified mouse models, theca cells isolated from normal cycling and PCOS ovaries, and state-of-the-art molecular and cellular technologies to accomplish three Specific Aims. Specific Aim 1: Determine the cellular and molecular consequences of disrupting Ar, Nr2f2 or Vcam1 selectively in the mouse theca/stromal cells in vivo and in human theca cells from normal cycling and PCOS women in culture. Specific Aim 2: Determine the molecular and cellular events and responses to androgens that are altered when AR, NR2f2 or Vcam1 is selectively over-expressed in mouse and human theca/stromal cells. Specific Aim 3: Determine the molecular mechanisms by which androgens induce Vcam1 in theca cells and if inflammatory factors and cells are theca critical for VCAM1 expression Results of these studies will provide key new insights into how androgens, AR, NR2F2 and VCAM1 impact ovarian dysfunction by determining what cellular events and molecular pathways are altered selectively in theca cells by different levels of AR receptor activation. By combining powerful mutant mouse models and well-characterized human theca cells, new strategies for regulating androgen action and the consequences of androgen excess in women will be established and provide for novel diagnostic markers and therapeutic targets for PCOS and hyperthecosis.